The present invention relates to recombinant parapoxviruses, to their preparation, and to vaccines and immunomodulators which contain them.
The novel, recombinantly altered parapoxviruses carry deletions and/or insertions in their genome. The deletion of segments of the genome of the parapoxviruses and/or the insertion of foreign DNA can lead to the reduction or loss of their pathogenicity (attenuation). Hereditary information from pathogens or biologically active substances is incorporated into the genome of the parapoxviruses by means of insertions. This foreign hereditary information is, as a constituent of the recombinant parapoxviruses, expressed, for example, in cell cultures, tissues or in intact organisms.
The recombinant parapoxviruses which have been prepared in accordance with the invention are employed, for example, in vaccines or immunomodulators. Expression of the foreign DNA in the genome of the parapoxviruses elicits, for example in a vaccinated individual, a defensive reaction against the pathogens which are represented by the foreign hereditary information. The non-specific resistances of the vaccinated individual can also be stimulated. (In that which follows, the term parapoxviruses is abbreviated to PPV).
PPV can themselves have an immunomodulatory effect since they stimulate non-pathogen-specific immune reactions in the organism. Thus, preparations of parapoxviruses are, for example, successfully employed in veterinary medicine for increasing general resistance.
While vaccines which have a pathogen-specific effect require several days to weeks, depending on the antigen, for establishing protection, they then provide long protection which lasts for months to years.
Consequently, vaccines which are prepared on the basis of recombinant parapoxviruses can be employed as biological products for the improved control of infectious diseases since they build up a long-lasting pathogen-specific immunity in the organism and also induce a non-pathogen-specific protection which sets in very rapidly.
The combination of the immunostimulatory properties of the PPV and the expression of foreign antigens which induce a homologous and/or heterologous pathogen-specific protection is novel. This permits the preparation of products which both mediate a rapid-onset, broad non-pathogen-specific protection against infections and also provide a long-lasting, pathogen-specific protection against infection.
The family of the vertebrate poxviruses (Chordopoxvirinae) is subdivided into individual, independent genera. The present invention relates to the genus of the PPV, which differ both structurally and genetically from the other poxviruses. The PPV are divided into three different species (Lit. #1):
Parapoxvirus ovis (also termed ecthyma contagiosum virus, contagious pustular dermatitis virus or orf virus), which is regarded as the prototype of the genus,
Parapoxvirus bovis 1 (also termed bovine papular stomatitis virus or stomatitis papulosa virus) and
Parapoxvirus bovis 2 (also termed udderpoxvirus, paravaccinia virus, pseudocowpox virus or milker""s nodule virus).
Parapoxvirus representatives which have been isolated from camels, red deer, chamois, seals and sealions have also been described. Whether these viruses are autonomous species within the parapoxvirus genus or whether they are isolates of the above-described species has still not been finally clarified.
Infections with PPV can elicit local diseases in both animals and man (zoonotic pathogens). Lit. #1 provides an overview of the syndromes which have so far been described. Prophylactic measures, such as vaccines, can be used to control the diseases. However, the activity of the vaccines which have thus far been obtainable, and which have been developed exclusively on the basis of Parapoxvirus ovis, is unsatisfactory (Lit. #2).
The invention relates to using PPV as a vector for foreign genetic information which is expressed.
Vectors based on avipox, racoonpox, capripox, swinepox or vaccinia virus have already been described as vectors for expressing foreign genetic information. The insights which have been gained in this connection cannot be transferred to PPV. As comparative investigations have demonstrated, there are morphological, structural and genetic differences between the individual genera of the poxviruses. Thus, serological methods can, for example, be used to differentiate the PPV from other poxvirus genera, a fact which is attributable to different protein patterns and to different hereditary information which is associated with this. For example, some representatives of the poxviruses have the ability to agglutinate erythrocytes. This activity is mediated by way of a surface protein, the so-called haemagglutinin (HA). PPV do not possess this activity.
Knowledge of the organization of the PPV genome is currently restricted to determinations of the size of the genome, the GC content of the nucleic acid, comparative restriction enzyme analyses, the cloning of individual genome fragments, and sequence analyses of part regions and the associated preliminary description of individual genes (for a review, see Lit. #1, Lit. #5, Lit. #6).
It is not currently possible to use insertion sites which are known in the case of vaccinia due to the fact that these sites are either lacking or have not been demonstrated in PPV.
Thus, attempts to identify the gene for thymidine kinase in the PPV genome and to use it as an insertion site, as in the case of the orthopoxviruses, were not successful. While Mazur and coworkers (Lit. #3) describe the identification of a segment of the PPV genome which they claim resembles the thymidine kinase gene of vaccinia virus (an orthopoxvirus), our own extensive investigations have not been able to confirm the existence of such a gene in PPV. Other authors (Lit. #1) have also not been able to find a thymidine kinase gene in PPV. The gene for HA is used as an insertion site for foreign DNA in vaccinia virus. As described above, PPV do not possess this activity.
In 1992, Robinson and Lyttle mentioned alternative insertion sites on the PPV genome (Lit. #1) without, however, providing a description or a precise characterization of these sites. There has furthermore still not been any description of the successful use of PPV as vectors.
In our own analytical investigations of the sequence of HindIII fragment I from PPV strain D1701, we found an ORF which possesses amino acid homology (36.1 to 38.3% identity; 52.8 to 58.6% similarity, GCG, Wisconsin Package 8.1, e.g. Pikup Program) with vascular endothelial growth factor (VEGF) from various mammalian species (e.g. mouse, rat, guinea pig, cow and man). Seq. ID No: 1 shows the nucleotide sequence of the gene in D1701, while Seq. ID No: 15 shows the amino acid sequence of the corresponding D1701 protein. Recently, a homologous gene was also described in PPV strains NZ2 and NZ7 (Lit. #6); however the function of this gene is not known. Other poxviruses, e.g. orthopoxviruses, are not known to have a corresponding gene. In the remainder of the text, this gene is termed VEGF gene.
Our sequence analysis of HindIII fragment I of D1701 led to the identification of another ORF which possesses homology with orthopoxvirus protein kinase genes and is known in vaccinia as F10L. The identity with the vaccinia F10L gene is 51% while the similarity is 70%. In the remainder of the application, this gene is termed PK gene. Seq. ID No: 2, No: 9 and No: 13 show versions of the nucleotide sequence of the gene in D1701, while Seq. ID No: 14 shows the amino acid sequence of the corresponding D1701 protein.
An additional ORF was found which overlaps the 3xe2x80x2 end of the PK gene and the 5xe2x80x2 end of the VEGF gene. Homology investigations showed that there was low identity (28%) and low similarity (51%) with the F9L gene in vaccinia. Seq. ID No: 5 and No: 10 show versions of the nucleotide sequence of the gene in D1701. In the remainder of the text, this gene is termed the F9L gene.
A further ORF, which, due to its similarity to a gene in PPV NZ2 (identity 76%, similarity 83%), is termed ORF3, was found within the ITR region. Seq. ID No: 4 shows the nucleotide sequence of the gene in D1701. In the remainder of the text, this gene is termed ORF3 gene.
The present invention relates to
1. Recombinantly prepared PPV having insertions and/or deletions.
2. Recombinantly prepared PPV having insertions and/or deletions in genome segments which are not required for virus multiplication.
3. Recombinantly prepared PPV having insertions and/or deletions in genome segments which are required for virus multiplication.
4. Recombinantly prepared PPV which contain insertions and/or deletions in the regions of HindIII fragment I from D1701 which are not expressed.
5. Recombinantly prepared PPV which contain insertions and/or deletions in the regions of HindIII fragment I from D1701 which are expressed.
6. Recombinantly prepared PPV according to 1 to 5, in which insertions and/or deletions are located in D1701 HindIII fragment I or in the DNA from other PPV which corresponds to this fragment.
7. Recombinantly prepared PPV which contain insertions and/or deletions in the region of the VEGF gene or adjoining this region.
8. Recombinantly prepared PPV which contain insertions and/or deletions in the region of the PK gene or adjoining this region.
9. Recombinantly prepared PPV which contain insertions and/or deletions in the region of the ITR segment or adjoining this region.
10. Recombinantly prepared PPV which contain insertions and/or deletions in the region of the HD1R gene or adjoining this region.
11. Recombinantly prepared PPV which contain insertions and/or deletions in the region of the F9L gene or adjoining this region.
12. Recombinantly prepared PPV which contain insertions and/or deletions in the region, or in the vicinity, of the gene which encodes the 10 kDa protein.
13. Recombinantly prepared PPV which contain insertions and/or deletions in the region of EcoRI fragment E from D1701, in which the gene encoding the 10 kDa protein is located.
14. Plasmid which contains HindIII fragment I from D1701 or DNA from other PPV which corresponds to this fragment.
15. Plasmid which contains HindIII fragment I from D1701 and which, in this fragment, contains deletions and/or insertions in regions which are required for virus replication.
16. Plasmid which contains HindIII fragment I from D1701 and which, in this fragment, contains deletions and/or insertions in regions which are not required for virus replication.
17. Plasmid which contains HindIII fragment I from D1701 and which, in this fragment, contains deletions and/or insertions in the regions which are not required for virus replication and which are not expressed.
18. Plasmid which contains HindIII fragment I from D1701 and which, in this fragment, contains deletions and/or insertions in the regions which are not required for virus multiplication and which lie in regions which are expressed.
19. Plasmid which contains HindIII fragment I from D1701 and which contains deletions and/or insertions in, or adjacent to, the VEGF gene of this fragment.
20. Plasmid which contains HindIII fragment I from D1701 and which contains deletions and/or insertions in, or adjacent to, the PK gene of this fragment.
21. Plasmid which contains HindIII fragment I from D1701 and which contains deletions and/or insertions in, or adjacent to, the ITR segment of this fragment.
22. Plasmid which contains HindIII fragment I from D1701 and which contains deletions and/or insertions in, or adjacent to, the HD1R gene and/or the F9L gene.
23. Plasmid which contains EcoRI fragment E from D1701 and which contains deletions and/or insertions in, or adjacent to, the gene which encodes the 10 kDa protein.
24. Plasmid which contains part of HindIII fragment I from D1701 in which deletions and/or insertions in accordance with 14 to 23 are present.
25. Plasmid according to 14 to 24, in which the DNA fragment from D1701 is replaced with a DNA from other PPV which corresponds to this fragment.
26. Plasmid according to 14 to 25, which either contains the whole of HindIII fragment I or only a part of it.
27. D1701 HindIII fragment I, or parts thereof, or fragments from other PPV which correspond to this fragment, having the sequence according to sequence listing ID No: 8 or No: 12.
28. DNA segment or parts of D1701 HindIII fragment I, or the segment from other PPV which corresponds to this segment, or parts thereof, which encodes VEGF protein in accordance with sequence listing ID No: 1.
29. DNA segment or parts of D1701 HindIII fragment I, or the segment from other PPV which corresponds to this segment or parts thereof, which encodes PK protein according to sequence listing ID No: 2, No: 9 or No: 13.
30. DNA segment, or parts thereof, for the HD1R gene having the sequence according to sequence listing ID No: 3 of PPV.
31. DNA segment, or parts thereof, for F9L having the sequence according to sequence listing ID No: 5 or ID No: 10 of PPV.
32. DNA segment, or parts thereof, for the ITR region having the sequence according to sequence listing ID No: 4 of PPV.
33. Gene products which have been prepared on the basis of the sequences of the DNA segments according to 27 to 32.
34. Recombinantly prepared PPV according to 1 to 13 which contain, as insertions, foreign DNA which encodes immunogenic constituents from other pathogens.
35. Recombinantly prepared PPV according to 1 to 13 and 34 which contain, as insertions, foreign DNA which encodes cytokines.
36. Process for preparing the viruses according to 1 to 13, 34 and 35, characterized in that the plasmids according to 14 to 26 are recombined with PPV in cells in the manner known per se and selected for the desired viruses.
37. Process for preparing the plasmids according to 23, characterized in that
1. a suitable PPV strain is selected,
2. its genome is purified,
3. the purified genome is treated with restriction enzymes,
4. the resulting fragments are inserted into plasmids, and
5. selection is carried out for the plasmids which contain the gene which encodes the 10 kDa protein, and
6. where appropriate, insertions and/or deletions are introduced into the gene encoding the 10 kDa protein,
7. the fragments described under 4 (above) can, where appropriate, also be prepared using alternative methods such as polymerase chain reaction (PCR) or oligonucleotide synthesis.
38. Process for preparing the plasmids according to 14 to 22 and 24 to 26, characterized in that
1. a suitable PPV strain is selected,
2. its genome is purified,
3. the purified genome is treated with restriction enzymes,
4. the resulting fragments are inserted into plasmids, and
5. selection is carried out for the plasmids which contain HindIII fragment I or fragments or constituents which correspond to this fragment,
6. and, where appropriate, insertions and/or deletions are introduced into these fragments in the resulting plasmids.
7. the fragments described under 4 (above) can, where appropriate, also be prepared using alternative methods such as polymerase chain reaction (PCR) or oligonucleotide synthesis.
39. Process for preparing D1701 HindIII fragment I or EcoRI fragment E, which encodes the 10 kDa protein, or the region from other PPV which corresponds to this fragment or segment, or parts thereof, characterized in that
1. a suitable PPV strain is selected,
2. its genome is purified,
3. the purified genome is treated with restriction enzymes,
4. and the desired fragments or segments are selected, or
5. where appropriate, the resulting fragments of the genome are initially inserted in plasmids and the plasmids containing the desired fragments are isolated, after which these plasmids are multiplied and the desired fragments are isolated from them.
6. the fragments described under 4 (above) can, where appropriate, also be prepared using alternative methods such as PCR or oligonucleotide synthesis.
40. Process for preparing the gene products according to 33, characterized in that the fragments obtainable in accordance with 39 are transferred into suitable expression systems and the genes are expressed using these systems.
41. Use of the recombinantly prepared PPV according to 1 to 13 in vaccines.
42. Use of the recombinantly prepared PPV according to 1 to 13 in products which both immunize and stimulate non-pathogen-specific immune defence.
43. Use of the recombinantly prepared PPV in immunomodulators which stimulate non-pathogen-specific immune defence.
44. Use of the recombinantly prepared PPV for heterologously expressing foreign DNA.
45. Use of the recombinantly prepared PPV as vectors for foreign DNA.
46. Use of the plasmids according to 14 to 16 for expressing parapox-specific genome segments.
47. Use of the plasmids according to 14 to 26 for preparing diagnostic agents.
48. Use of the genome fragments according to 27 to 32 for preparing diagnostic agents.
49. DNA segments according to sequence listing ID No: 6 (promoter of the VEGF gene).
50. Use of the DNA segment according to 49 as a promoter for expressing DNA.
The above-described genome fragments of PPV, which can be inserted into plasmids or viruses and which can be present as free DNA segments, encompass the given DNA sequences and their variants and homologs.
The above-listed terms have the following meanings:
Attenuation is a
process in which, as a result of an alteration to their genome, the PPV have become less pathogenic or not pathogenic, or less virulent or not virulent, for animals or man.
Deletions are
pieces of DNA which are missing from the PPV genome.
Deletion plasmids are
plasmids which, in addition to the plasmid DNA, carry segments of the PPV genome from which pieces have been removed.
Genome segments which are necessary (essential) for virus multiplication are
parts of the whole PPV genome which are indispensable for the in-vitro multiplication of PPV, i.e. are indispensable for forming infectious virus progeny.
Interference with genes which are essential for virus multiplication leads to the virus multiplication being interrupted. If, for example, parts of one of these genes, or the entire gene, is removed, replication of the virus terminates at a defined point in the multiplication cycle of the virus. Infection or treatment with mutants of this nature do not lead to any release of infectious progeny from the animal. If parts of an essential gene, or a whole essential gene, is/are replaced with foreign DNA, or if foreign DNA is inserted into essential genes, it is possible to construct vector vaccines which are unable to multiply in the vaccinated individual and which are consequently not excreted as infectious pathogens.
Genome segments which are not required (nonessential) for virus multiplication are
parts of the whole PPV genome which can be dispensed with for the in-vitro multiplication of PPV, i.e. for forming infectious virus progeny.
Foreign DNA elements (foreign DNA) are
DNA pieces, e.g. foreign genes or nucleotides sequences, which are not originally present in the PPV which is employed in accordance with the invention.
Foreign DNA is inserted into the PPV for the following reasons:
1. for expressing the foreign DNA
2. for inactivating functions of pieces of the PPV DNA
3. for labelling the PPV.
Depending on these reasons, different foreign DNA is inserted. If foreign DNA is to be expressed in accordance with (1), the inserted foreign DNA will at least carry an open reading frame which encodes one or more desired foreign protein(s). Where appropriate, the foreign DNA additionally contains its own or foreign regulatory sequences. The capacity for taking up foreign DNA can be increased by creating deletions in the genome of the virus. In general, the length is between 1 nucleotide and 35,000 nucleotides, preferably between 100 and about 15,000 nucleotides.
Examples which may be mentioned are genes, or parts of genes, from viruses such as
Herpesvirus suid 1,
Equine herpesviruses
Bovine herpesviruses
Foot and mouth disease virus,
Bovine respiratory syncytial virus,
Bovine parainfluenza virus 3,
Influenza virus
Calicivirus
Flaviviruses, e.g. bovine virus diarrhoea virus or classical swine fever virus
or of bacteria, such as
Pasteurella spec.,
Salmonella spec.,
Actinobacillus spec.,
Chlamydia spec.,
or of parasites, such as
Toxoplasma,
Dirofilaria,
Echinococcus.
If foreign DNA is to be inserted in accordance with (2), the insertion of a suitable foreign nucleotide is in principle sufficient for interrupting the DNA sequence of the vector virus. The maximum length of the foreign DNA which is inserted for the inactivation depends on the capacity of the vector virus to take up foreign DNA. In general, the length of the foreign DNA is between 1 nucleotide and 35,000 nucleotides, preferably between 100 and 15,000 nucleotides, particularly preferably between 3 and 100 nucleotides.
If DNA sequences are to be inserted for labelling in accordance with (3), their length depends on the detection method used for identifying the labelled virus. In general, the length of the foreign DNA is between 1 and 25,000 nucleotides, preferably between 20 nucleotides and 15,000 nucleotides, particularly preferably between 5 and 100 nucleotides.
Gene library
is the entirety of the fragments of a genome which are contained in vectors which are capable of replication. The library is obtained by fragmenting the genome and inserting all the fragments, a few of the fragments or a major part of the fragments into vectors which are capable of replicating, for example plasmids.
A genome fragment is
a piece of a genome which can be present in isolated form or can be inserted into a vector which is capable of replicating.
Inactivation by insertion means
that the inserted foreign DNA prevents the native PPV genome sequences from being expressed or from functioning.
Insertions are
pieces of DNA which have been additionally incorporated into the PPV genome. Depending on the reason for the insertion, the length of the DNA pieces can be between 1 nucleotide and several thousand nucleotides (see definition of xe2x80x9cforeign DNAxe2x80x9d as well).
Insertion plasmids are
plasmids, in particular bacterial plasmids, which contain the foreign DNA to be inserted flanked by PPV DNA sequences.
Insertion sites are
sites in a viral genome which are suitable for receiving foreign DNA.
Cloning means
that the PPV genomic DNA is isolated and fragmented. The fragments, or a selections of the fragments, is/are then inserted into customary DNA vectors (bacterial plasmids or phage vectors or eukaryotic vectors).
Lit. #11 provides a selection of methods for preparing and cloning DNA fragments. The DNA vectors, containing the PPV DNA fragments as inserts, are used, for example, for preparing identical copies of the originally isolated PPV DNA fragments.
Labelling by insertion means
that the inserted foreign DNA enables the modified PPV to be subsequently identified.
ORF (open reading frame) is understood as meaning a sequence of nucleotides, at the DNA level, which defines the amino acid sequence of a potential protein. It consists of a number, which is determined by the size of the protein which it defines, of nucleotide triplets which is delimited at the 5xe2x80x2 end by a start codon (ATG) and at the 3xe2x80x2 end by a stop codon (TAG, TGA or TAA).
Regulatory sequences are
DNA sequences which exert an effect on the expression of genes. Sequences of this nature are known from Lit. #15.
Preference may be given to mentioning the VEGF promoter as described in sequence listing ID No: 6.
Recombinant PPV are
PPV having insertions and/or deletions in their genome. In this connection, the insertions and deletions are prepared using molecular biological methods.
Repetitive (DNA) sequences are
identical nucleotide sequences which occur in the PPV genome either directly one after the other or scattered at different sites.
Vector virus is
a PPV which is suitable for the insertion of foreign DNA and which can transport the inserted foreign DNA, in its genome, into infected cells or organisms, and which, where appropriate, enables the foreign DNA to be expressed.
The novel PPV according to 1 to 12 (above) are prepared as follows:
1. Selection of a suitable PPV strain
2. Identification of genome segments in the PPV genome which possess insertion sites
2.a Identification of PPV genome segments possessing insertion sites in genes which are non-essential for virus multiplication,
2.b Identification of PPV genome segments possessing insertion sites in genes which are essential for virus multiplication,
2.c Identification of genome segments possessing insertion sites in regions outwith genes in the PPV genome and/or in gene duplications
2.d Other methods for identifying genome segments possessing insertion sites
2.e Demands placed on an insertion site
2.1 Identification of insertion sites
2.1.1 Purification of the PPV genome
2.1.2 Cloning the genome fragments and establishing a gene library
2.1.3 Sequencing for the purpose of identifying genes or genome segments outwith genes
2.1.4 Selection of the clones containing PPV genome fragments for further processing
2.2 ITR region, VEGF gene, PK gene, gene encoding the 10 kDa protein, and the region between the PK gene and the HD1R gene, as insertion sites
2.2.1 Cloning the VEGF gene
2.2.2 Cloning the protein kinase gene
2.2.3 Cloning the gene region which encodes the 10 kDa protein
2.2.4 Cloning the ITR region (inverted terminal repeat region) or the genome segment which lies between the PK gene and the HD1R gene
3. Construction of insertion plasmids or deletion plasmids which contain the foreign DNA to be inserted,
3.1 Identifying or preparing restriction enzyme recognition sites which only occur once, i.e. unique restriction sites, in the cloned genome fragments and inserting foreign DNA
3.2 Deleting genome sequences in the cloned genome fragments and inserting foreign DNA
3.3 A combination of #3.1 and #3.2
4. Construction of a recombinant PPV in accordance with 1 to 12 (above).
1. Selection of a suitable PPV strain
In principle, all PPV species are suitable for implementing the present invention. Virus strains are preferred which can be multiplied to titres  greater than 105 PFU (plaque forming unit)/ml in a tissue culture and which can be prepared in pure form as extracellular, infectious virus, from the medium of the infected cells. The species from the PPV genus which may be mentioned as being prefer red is PPV ovis (orf viruses).
The strain of PPV ovis which may be mentioned as being particularly preferred is D1701, which was deposited on 28.04.1988, in accordance with the Budapest Treaty, at Institut Pasteur, C.N.C.M. under Reg. No. CNCM I-751, and also its variants and mutants.
The viruses are multiplied in a customary manner in tissue cultures of animal cells such as mammalian cells, e.g. in sheep cells or bovine cells, preferably in bovine cells such as the permanent bovine kidney cell line BK-K1-3A (or its descendants) or monkey cells, such as the permanent monkey kidney cells MA104 or Vero (or their descendants).
The multiplication is effected, in a manner known per se, in stationary, roller or carrier cultures in the form of compact cell aggregates or in suspension cultures.
The cells or cell lawns which are used for multiplying the viruses are multiplied virtually to confluence or up to optimal cell density in a customary manner. The cells are infected with virus dilutions which correspond to an MOI (=multiplicity of infection, corresponds to infectious virus particles per cell).
The viruses are multiplied with or without the addition of animal sera. When serum is employed, it is added to the multiplication medium at a concentration of 1-30 vol %, preferably 1-10 vol %.
Infection and virus multiplication are carried out at temperatures of between room temperature and 40xc2x0 C., preferably between 32 and 39xc2x0 C., particularly preferably at 37xc2x0 C., over several days, preferably until the infected cells have been completely destroyed. In association with harvesting the virus, virus which is still cell-bound can additionally be released mechanically or by means of ultrasonication or by means of mild enzymic proteolysis, for example using trypsin.
The virus-containing medium from the infected cells can then be worked up further, for example by removing the cell debris by means of filtration using pore sizes of, for example, 0.2-0.45 xcexcm and/or low-speed centrifugation.
Filtrates or centrifugation supernatants can be used for virus enrichment and purification. For this, filtrates or supernatants are subjected to high speed centrifugation until the virus particles sediment. Where appropriate, further purification steps can follow, for example by means of centrifugation in a density gradient.
2. Identification of genome segments possessing insertion sites in the PPV genome
Various regions of the PPV genome can be used as insertion sites when inserting foreign DNA. Foreign DNA can be inserted
a. into genes which are non-essential for virus multiplication in vitro and/or in vivo,
b. into genes which are essential for virus multiplication, and/or
c. in regions which do not possess any gene function.
2.a Identification of genome segments in the PPV genome which possess insertion sites in genes which are not essential for virus multiplication
i. Viral genes which are not essential for multiplication of the virus are found, for example, by means of carrying out comparative investigations using representatives of different PPV species. Genes which do not occur in one or more isolates or strains of a PPV species but which are found in other isolates or strains are potentially non-essential.
ii. Genes which are not essential for virus multiplication can also be identified in an alternative manner. After PPV genome fragments, which may, for example, be present as cloned fragments, have been sequenced, these DNA sequences are examined for possible xe2x80x9copen reading framesxe2x80x9d (ORF). If an ORF is found, the function of this ORF as a gene is verified by demonstrating transcription and/or translation. In order to establish whether the gene which has been found is non-essential for virus multiplication, molecular genetic methods are used to remove the gene from the PPV genome, or to destroy it partially or to interrupt it by means of introducing (a) mutation(s), and the ability of the resulting virus to multiply is then investigated. If the virus is able to replicate even without the existence of the manipulated gene, the latter is a non-essential gene.
Examples of identified insertion sites
i. The VEGF gene of PPV ovis may be mentioned at this point as an example of a non-essential PPV gene which can be used as an insertion site for foreign DNA. This gene is found in the Parapoxvirus ovis strains (NZ-2, NZ-7 and D1701) which have been investigated (Lit. #6). This VEGF gene has not been demonstrated in some PPV strains, for example representatives of PPV bovis 1. The region on the PPV genome containing the VEGF gene can be identified with the aid of the DNA sequence shown in sequence listing ID No: 1. The customary methods of molecular biology can be used to find the gene on the genome of a PPV by means of hybridization experiments, genome sequence analyses and/or polymerase chain reactions.
ii. The gene for the 10 kDa PPV protein may be mentioned as another example of a potentially non-essential PPV gene. This gene is found in strains of Parapoxvirus ovis (NZ-2, NZ-7 and D1701). Customary methods of molecular biology can be used to identify the region on the PPV genome which contains the gene for the 10 kDa protein, for example by means of polymerase chain reactions (PCR). Lit. #8 gives the DNA sequence of the 10 kDa protein gene. The primers which can be used for a PCR are specified, for example, in Lit. #8. Sequence listing ID No: 11 shows the DNA sequence of the 10 kDa-specific PCR product from D1701.
For the purpose of inserting foreign DNA, the non-essential gene can be removed from the PPV genome either in parts or entirely. However, it is also possible to insert foreign DNA into the non-essential gene without removing any regions of the PPV gene. Restriction enzyme recognition sites can, for example, be used as insertion sites.
2.b. Identification of PPV genome segments, on the PPV genome, which possess insertion sites in genes which are essential
i. Essential genes can be identified by sequencing viral genome fragments, which are, for example, present as cloned fragments, and then identifying possible ORFs.
If an ORF is found, its function as a gene is verified by demonstrating transcription and/or translation. In order to establish whether the gene has been found is essential for virus multiplication, molecular genetic methods are used to destroy the gene in the PPV genome, for example by removing parts of the gene, or the entire gene, or by means of inserting foreign DNA, and then investigating the ability of the resulting virus to multiply.
If the resulting virus mutant is unable to replicate, the gene is then very probably an essential gene.
If the virus mutant is only able to grow on complementing cell lines, this then proves that the gene is essential.
Examples of insertion sites
The protein kinase gene (PK gene) of PPV D1701 may be mentioned as an example. The PK gene is expressed late in the multiplication cycle of the virus. The versions of the DNA sequence shown in sequence listing ID No: 2, No: 9 or No: 13 can be used to identify the PK gene on the PPV genome. The customary methods of molecular biology can be used to find the region containing the gene, for example by means of hybridization experiments, genome sequence analyses and/or polymerase chain reactions.
For the purpose of inserting foreign DNA, the essential gene can be removed, either in parts or entirely, from the PPV genome. However, it is also possible to insert the foreign DNA into the essential gene without removing regions from the PPV gene.
2.c Identification of genome segments on the PPV genome possessing insertion sites in regions outwith genes and/or in gene duplications
Genome segments which do not encode functional gene products and which do not possess any essential regulatory functions (so-called intergenic segments) are, in principle, suitable for use as insertion sites for foreign DNA. Regions containing repetitive sequences are particularly suitable, since changes in parts of a region can be offset by sequence repetitions which remain. Genes which occur in two or more copies, so-called gene duplications, also come within this category.
Genes in the ITR region or in duplicated segments of the PPV genome exist in two copies in the viral genome. After one copy of such a gene has been removed or altered, and foreign DNA has been inserted, stable PPV recombinants can be obtained even if the altered gene is important for virus multiplication. A second, unaltered gene copy may be adequate for the function of the gene.
i. Sequence analyses of the PPV genome are used to identify genome sequences which do not encode gene products. Genome regions which do not exhibit either an ORF after sequence analysis or virus-specific transcription and which do not possess any regulatory function represent potential insertion sites. In particular, the cleavage sites for restriction enzymes in these regions represent potential insertion sites. In order to check whether a suitable insertion site is present, known molecular biological methods are used to insert foreign DNA into the potential insertion site, and the viability of the resulting virus mutant is then investigated. If the virus mutant which carries foreign DNA in the possible insertion site is capable of multiplication, the site being investigated is a suitable insertion site.
ii. DNA hybridization experiments and/or sequence analyses are used to identify repetitive sequences and gene duplications. In the hybridization experiments, cloned or isolated genome fragments from a PPV are used as probes for hybridizations with fragments of PPV DNA. The genome fragments of the PPV which hybridize with more than one fragment of the total PPV genome contain one or more repetitive sequences. In order to locate the repetitive sequence or the duplicated genome regions accurately on the genome fragment, the nucleotide sequence of this fragment is determined. In order to establish whether a potential insertion site is a suitable insertion site in the whole PPV genome, foreign DNA has to be inserted into a repetitive sequence or into a copy of the gene duplication and the PPV genome fragment containing the insert has to be incorporated into the viral genome. The ability of the recombinant virus containing the foreign DNA to multiply is then examined. If the recombinant virus multiplies, the identified recognition site is suitable as an insertion site.
Examples of insertion sites
i. The genome segment between the gene for the protein kinase and the HD1R gene (sequence listing ID No: 7) may be mentioned as an example of an intergenic region.
ii. The ITR region (sequence listing ID No: 4) may be mentioned as an example of a repetitive sequence.
iii. The potential gene xe2x80x9cORF 3xe2x80x9d (in the ITR region) and the VEGF gene may be mentioned as examples of gene duplications in PPV strain D1701. Hybridization studies demonstrated that a region containing the VEGF gene has been duplicated in the present strain D1701 and translocated to the other end of the virus genome, so that two copies of the VEGF gene are present.
With the aid of the sequences in sequence listing ID No: 4 (ITR sequence with xe2x80x9cORF3xe2x80x9d gene) and ID No: 7 (region between PK and HD1R genes), customary methods of molecular biology, such as hybridization experiments, genome sequence analyses and/or polymerase chain reactions, can be used to find the corresponding genome regions in other PPV.
2.d Other methods for identifying insertion sites
In general, modifications of the viral genome sequences can also be used to find possible insertion sites on the PPV genome. Genome sites at which nucleotide substitutions, deletions and/or insertions, or combinations thereof, do not block virus multiplication constitute possible insertion sites. In order to check whether a potential insertion site is a suitable insertion site, known molecular biological methods a re used to insert foreign DNA into the potential insertion site and the viability of the resulting virus mutant is investigated. If the virus recombinant is able to multiply, the site under investigation is a suitable insertion site.
2.1 Identification of insertion sites
2.1.1 Purification of the PPV genome
For the purpose of cloning PPV insertion sites by means of molecular genetics, the PPV genome is first of all purified. The genome is isolated from the virus prepared in accordance with 1 (above) and then purified. Native viral DNA is preferably extracted by treating the purified virions with aqueous solutions of detergents and proteases.
Detergents which may be mentioned are anionic, cationic, amphoteric and nonionic detergents. Preference is given to using ionic detergents. Sodium dodecyl sulphate (sodium lauryl sulphate) is particularly preferred.
Proteases which may be mentioned are all proteases which function in the presence of detergents, such as proteinase K and pronase. Proteinase K may be mentioned as being preferred.
Detergents are employed in concentrations of 0.1-10 vol %, with 0.5-3 vol % being preferred.
Proteases are employed in concentrations of 0.01-10 mg/ml of virus lysate, with 0.05-0.5 mg/ml of virus lysate being preferred.
Preference is given to carrying out the reaction in an aqueous buffered solution in the presence of DNase inhibitors. Buffering substances which may be mentioned are: salts of weak acids with strong bases, e.g. tris(hydroxymethylaminomethane), salts of strong acids with weak bases, e.g. primary phosphates, or mixtures thereof.
The following buffer system may be mentioned as being preferred: tris(hydroxymethylaminomethane).
The buffering substances or buffering systems are employed at concentrations which ensure pH values at which the DNA does not denature. Preference is given to pH values of 5-9, with particular preference being given to values of 6-8.5 and very particular preference being given to values of 7-8; operating in the neutral range may be mentioned, in particular.
An example of a DNase inhibitor is ethylenediaminetetraacetic acid at concentrations of 0.1-10 mM (millimole), with approx. 1 mM being preferred.
After that, the lipophilic components of the virus lysate are extracted. Solvents such as phenol, chloroform, isoamyl alcohol, or their mixtures, are used as extracting agents. Preference is given to using a mixture of phenol and chloroform/isoamyl alcohol initially, with the extraction taking place in one or more stages.
Examples of other methods for isolating virus DNA are centrifugation of a virus lysate in a CsCl density gradient or gel electrophoresis (see Lit. #14).
The extraction of nucleic acids is described in Lit. #13.
The DNA which has been extracted in this way is preferably precipitated from the aqueous solution with, for example, alcohol, preferably with ethanol or isopropanol, and in the added presence of monovalent salts such as alkali metal chlorides or acetates, preferably lithium chloride, sodium chloride, or sodium acetate or potassium acetate (see loc. cit.).
2.1.2 Cloning the genome fragments
The viral DNA which has been purified in this way is now used to prepare DNA fragments. For this, it is, for example, treated with restriction enzymes. Examples of suitable restriction enzymes are EcoRI, BamHI, HindIII and KpnI. Alternatively, genome fragments can be synthesized by means of the polymerase chain reaction (PCR). For this, primers are selected from sequence segments of the viral genome which are already known, and the genome segment which is delimited by the primer pair is synthesized in vitro using, for example, Taq polymerase or Pfu polymerase.
The DNA fragments resulting from restriction digestion or PCR can be cloned into vector systems using the methods described in (Sambrook 89). For example, depending on the size of the DNA fragment to be cloned in each case, plasmid vectors, lambda phage vectors or cosmid vectors are available for this purpose.
2.1.3 Sequencing, identifying and characterizing genes, and verifying their expression
Genome fragments which are cloned into vectors are first of all analyzed by sequencing. The inserted DNA fragments are mapped using different restriction enzymes and suitable subfragments are cloned into plasmid vectors. The sequencing reaction is effected, for example, using the T7 Sequencing Kit supplied by Pharmacia in accordance with the manufacturer""s instructions. The double-stranded plasmid DNA which is required for this is preferably prepared using the PEG method (Hattori and Sakaki 1985). xe2x80x9cOpen reading frames (ORF)xe2x80x9d which are present in the genome fragments are identified by means of computer analysis (GCG, see above). Information about the respective function of the identified ORFs can be obtained by means of comparing their sequences with other gene sequences of known function which are contained in a database. The identified ORFs are functionally characterized by detecting their respective corresponding transcripts in virus-infected cells. For this, the AGPC method (Chomczynski and Sacchi, (20) 1987) is, for example, used to isolate the total RNA from virus-infected cells. The specific transcripts, and their 5xe2x80x2 and 3xe2x80x2 ends, can then be identified by means of Northern blot analysis or primer extension and RNA protection experiments. As an alternative, it is possible to express the virus protein which is encoded by an identified ORF in vitro, then to use the expression product to obtain antisera and to use these antisera to demonstrate expression of the ORF.
In order to establish whether the gene which has been found is non-essential for virus multiplication, the gene can be destroyed by means of gene disruption or gene deletion. In this context, either the entire gene, or parts of it, are removed from the PPV genome or the reading frame of the gene is interrupted by inserting foreign gene sequences. The ability of the resulting virus to multiply is examined. If the virus can replicate even without the existence of the destroyed gene, this gene is then a non-essential gene.
2.1.4 Selecting the clones containing PPV genome fragments
Which of the PPV genome fragment-containing clones obtained above are employed depends on whether the recombinant PPV which are to be prepared are to be (i) capable of replication or (ii) defective in their replication.
i. If recombinant PPV are to be prepared which are capable of replication despite the insertion and/or deletion, further processing is carried out on cloned viral genome fragments which contain genes or genome regions outwith genes which are non-essential for virus multiplication or which contain gene duplications.
Virus mutants are used to test whether the gene or genome region to hand is a non-essential region of the virus genome or a gene duplication. For this, molecular biological methods are used to inactivate the gene or genome region in the PPV which is being investigated, for example by partially or completely deleting the region in question, and the ability of the virus mutant to multiply is examined. If the virus mutant can multiply despite the gene or genome region in question having been inactivated, the gene or genome region under investigation is a non-essential region.
Preference is given to cloned genome fragments of the PPV which contain complete versions of the non-essential genes. In addition to this, the flanking viral genome regions at both ends of the genes or the genome regions should also be present. The length of the flanking regions should be more than 100 base pairs. If such genome clones are not available, they can be prepared from existing gene clones by means of molecular biological methods. If the cloned genome fragments additionally contain genome regions which are not required for the present preparation, these regions can be removed by means of subclonings.
ii. If recombinant PPV are to be prepared which have lost the ability to form infectious progeny as a result of the insertion and/or deletion, cloned viral genome fragments which contain genes or genome regions outwith genes which are essential for virus multiplication are subject to further processing.
Virus mutants can be used to test whether the gene or genome region to hand is an essential region of the virus genome. For this, molecular biological methods are used to inactivate the gene or the genome region in the PPV under investigation, for example by partially or completely deleting the region in question, and the ability of the virus mutant to multiply is examined. If the virus mutant can no longer multiply as a result of the gene or genome region in question having been inactivated, the gene or genome region under investigation is an essential region.
Preference is given to cloned genome fragments of the PPV which contain complete versions of the essential genes. In addition to this, the flanking viral genome regions should likewise be present at both ends of the genes or the genome regions. The length of the flanking regions should be more than 100 base pairs. If such genome clones are not available, they can be prepared from existing genome clones by means of molecular biological methods. If the cloned genome fragments contain additional genome regions which are not required for the present preparation, these regions can be removed by means of subclonings.
2.2 ITR region, VEGF gene, PK gene, gene encoding the 10 kDa protein, and the region between the PK gene and the HD1R gene, as insertion sites
If the ITR region, the VEGF gene, the PK gene, the gene which encodes the 10 kDa protein, or the intergenic region between the PK gene and the HD1R gene, is to be used as an insertion site in a PPV, the corresponding regions of the PPV genome, which contain the insertion sites, have to be isolated. For this, the corresponding regions of the PPV genome are cloned.
2.2.1 Cloning the VEGF gene
The gene which encodes VEGF is located on the PPV genome and is then isolated in parts or in its entirety together with its flanking genome segments. For this, the PPV is preferably multiplied in accordance with #1 and the genome is purified in accordance with #2.1.1.
a. The VEGF gene is preferably amplified by means of a polymerase chain reaction (PCR). The start sequences (primers) which are required for this reaction are derived from the DNA sequence of the VEGF gene which is depicted in sequence listing ID No: 1. The resulting amplificate is then preferably cloned.
b. The region which contains the VEGF gene and its flanking genome segments is preferably obtained by fragmenting the PPV genome and isolating and cloning the corresponding genome fragment(s). For this, the purified genome of the virus is cleaved as described in #2.1.2, preferably using the restriction enzyme HindIII. The genome fragments which are obtained after the enzyme digestion are preferably fractionated by means of electrophoretic or chromatographic methods in order to identify the genome fragment(s) which carries/carry the VEGF gene and its flanking genome segments.
Electrophoretic fractionations in agarose or polyacrylamide are carried out using standard methods which are described in
Current Protocols in Molecular Biology 1987-1988, Wiley-Interscience, 1987.
A Practical Guide to Molecular Cloning, Perbal, 2nd edition, Wiley Interscience, 1988
Molecular Cloning, loc. cit.
Virologische Arbeitsmethoden [Practical Methods in Virology], Volume III, Gustav Fischer Verlag, 1989.
The genome fragments which carry the VEGF gene and its flanking sequences are identified, for example, by means of hybridization with defined nucleic acid probes. For this, the fractionated genome fragments are transferred to filters and hybridized with VEGF-specific, labelled nucleic acid probes in accordance with the Southern blot method. The methods for transferring the genome fragments and for the hybridization can be carried out in accordance with standard protocols, as described under xe2x80x9cSouthern Blottingxe2x80x9d in Molecular Cloning loc. cit. The oligonucleotides or nucleic acid fragments which can be used as probes can be derived from sequence listing Seq ID No: 1. For example, the TaqI subfragment (366 bp), which can be identified by means of Seq ID No: 1, is employed as a hybridization probe.
The genome fragments which have been demonstrated to contain parts, or preferably the whole, of the VEGF gene and the flanking genome segments, are isolated and cloned. The appropriate genome fragment(s) is/are electrophoretically isolated, for example, from the appropriate region of the gel by means of electroelution or by using the low-melting agarose method.
In order to clone the VEGF gene, the genome fragments which have been prepared above are inserted into bacterial or eukaryotic vectors. Plasmid or phage vectors are particularly preferred initially. In order to insert the genome fragment, double-stranded plasmid or phage vector DNA molecules are treated with restriction enzymes so that suitable ends are produced for the insertion.
Known plasmids, such as pBR322 and its derivatives, e.g. pSPT18/19, pAT153, pACYC 184, pUC18/19 and pSP64/65, are used as plasmids.
The known variants of phage lambda, such as phage lambda ZAP and phage lambda gt10/11, or phage M13mp18/19, are, for example, used as phage vectors.
The restriction enzymes which can be used are known, for example, from Gene volume 92 (1989) Elsevier Science Publishers BV Amsterdam.
The plasmid or the phage vector which has been treated with restriction enzyme is mixed with an excess of the DNA fragment to be inserted, for example in an approximate ratio of 5:1, after which the mixture is treated with DNA ligase to ligate the fragment into the vector. In order to propagate the plasmid or phages, the ligation mixture is introduced into prokaryotic or eukaryotic cells, preferably into bacteria (e.g. Escherichia coli strain K12 and its derivatives) and the latter are replicated.
The bacteria are transformed and selected as described in Molecular Cloning loc. cit.
The identity of the foreign DNA is preferably verified by means of hybridization experiments and particularly preferably by means of sequence analyses. Subclonings are performed where appropriate.
2.2.2 Cloning the protein kinase gene
The gene which encodes the protein kinase is located on the PPV genome and then isolated in parts or in its entirety together with its flanking genome segments.
As described for the cloning of the VEGF gene, this region can be isolated by fragmenting the PPV genome (cleavage sites, see FIG. 1), preferably followed by cloning the fragments and selecting the fragments or clones which contain parts of the PK gene or, preferably, the entire PK gene together with flanking DNA sequences of the PPV genome. DNA molecules which can be employed as primers for a PCR or as probes for a hybridization can be derived from sequence listing ID No: 2 or ID No: 9.
Subclonings are performed where appropriate.
2.2.3 Cloning the gene which encodes the 10 kDa protein
The gene which encodes the 10 kDa protein is located on the PPV genome using the method described (above) in detail for the VEGF gene and the PK gene and isolated in parts or, preferably, in its entirety together with its flanking PPV genome sequences.
In this case, the region of the PPV genome which contains the gene for the 10 kDa protein, or parts thereof, is obtained, preferably by means of PCR and/or cloning and identifying and selecting the suitable clones.
Lit. #8 provides details of DNA molecules which can be employed as primers for a PCR or as probes for a hybridization. Subclonings are performed where appropriate.
2.2.4 Cloning the inverted terminal repeat region for the genome segment which lies between the PK gene and the HD1R gene
The approach corresponds to the cloning of the VEGF gene which has been described in detail (above). The DNA molecules which can be employed, as primers for a PCR or as probes for a hybridization, for isolating the appropriate regions are evident from sequencing listing ID No: 4 (ITR region) and No: 7 (region between the PK gene and the HD1R gene).
3. Construction of insertion plasmids or deletion plasmids
So-called insertion plasmids, which can be used for inserting foreign DNA into the PPV genome, are prepared on the basis of the PPV genome fragments which are identified, located and cloned as described in Section 2. The insertion plasmids carry the foreign DNA which is to be inserted into the PPV, flanked by segments of the PPV genome. There are various options for preparing insertion plasmids: the following may be mentioned here as examples:
3.1 Identification or preparation of unique restriction enzyme recognition sites in the cloned genome fragments which are obtained as described in 2.1.4 or 2.2, and insertion of foreign DNA
Restriction cleavage sites which only occur once, i.e. are unique, can, for example (see 2.1.3), be identified in the PPV nucleotide sequences which have been determined.
Synthetically prepared oligonucleotides which carry new unique cleavage sites for restriction enzymes can be incorporated into these unique restriction sites.
The resulting plasmids are propagated and selected as described previously.
Alternatively, PCR can be used, as described by Jacobs et al. (Lit. #12), to incorporate new unique restriction enzyme recognition sites into the PPV genome fragments.
The unique restriction enzyme recognition sites which have been identified and/or prepared are used for inserting foreign DNA into the PPV genome.
The foreign DNA is inserted using known methods (Lit. #11).
3.2 Deletion of genome sequences in the cloned genome fragments and insertion of foreign DNA
Subfragments can, for example, be deleted from the cloned PPV genome fragments by treating the latter with restriction enzymes which preferably possess more than one, particularly preferably 2, recognition sites. After the enzyme treatment, the resulting fragments are fractionated as described above, for example electrophoretically, and isolated, and the appropriate fragments are joined together once again by means of ligase treatment. The resulting plasmids are propagated and the deleted plasmids are selected.
Alternatively, a unique restriction enzyme recognition site on the PPV genome fragment is used as the starting point for bidirectionally degrading the fragment with an endonuclease, for example the enzyme Bal31. The size of the deletion can be determined by the period during which the enzyme acts and can be checked by means of gel electrophoresis. Synthetic oligonucleotides are ligated to the newly produced fragment ends as described under 3.1 (above).
The foreign gene is transferred into the PPV genome in only a small percentage of the entire PPV population.
For this reason, selection systems are required to separate recombinant PPV from wild-type PPV (Lit. #16).
Preference is given to using the gpt selection system, which is based on the E. coli guanyl-phosphoribosyl transferase gene. When expressed in a eukaryotic cell, this gene confers resistance to mycophenolic acid, which is an inhibitor of purine metabolism. Its use in the construction of recombinant vector viruses has been described many times (see Lit. #16/#17).
4. Construction of a recombinant PPV in accordance with 1 to 12
Foreign DNA is inserted into the PPV genome by:
a. simultaneously transfecting the DNA of the insertion or deletion plasmid and infecting with PPV in suitable host cells,
b. transfecting the DNA of the insertion or deletion plasmid and then infecting with the PPV in suitable host cells,
c. infecting the PPV and then transfecting with the DNA of the insertion or deletion plasmid in suitable host cells.
The methods for the procedures which are suitable for this purpose are known. The transfection can be effected using known methods such as the calcium phosphate technique, liposome-mediated transfection or electroporation (see Lit. #18).
1. Infection with PPV:
Cell cultures which permit good virus multiplication and efficient transfection, for example the permanent bovine kidney cell line BK-K1-3A, are preferred for preparing PPV containing foreign DNA.
2. Preparation of the insertion or deletion plasmid DNA:
The transformed cells, for example bacteria, which were obtained by the previously described methods and which harbour the insertion or deletion plasmids are propagated and the plasmids are isolated from the cells in a known manner and subjected to further purification. The purification is effected, for example, by means of isopycnic centrifugation in a density gradient of, for example, CsCl or by means of affinity purification on commercially obtainable silica particles.
3. Transfection:
Purified circular or linearized plasmid DNA is preferably used for the transfection. The purification is effected as indicated under section 2 (above), for example.
4. Culturing transfected and infected cells
The cells are cultured using the above-described methods. When a cytopathic effect appears, the culture medium is removed, where appropriate freed from cell debris by centrifugation or filtration and, where appropriate stored, and also worked up using the conventional methods for the single-plaque purification of viruses.
The following method is employed when preparing recombinant PPV:
BK-KL-3A cells which have grown to confluence are infected with an infection dose having an MOI (multiplicity of infection) of from 0.001 to 5, preferably of 0.1. Two hours later, the infected cells are transfected, for example, with the DNA (2-10 xcexcg) of the plasmid pMT-10, either using the CaPO4-glycyerol shock method or using a Transfection Kit in accordance with the manufacturer""s instructions (DOSPER, Boehringer-Mannheim). These cell cultures are then incubated with medium at 37xc2x0 C. and under a 5% CO2 atmosphere for from three to six days until a cpe or plaque formation becomes visible.
Depending on the inserted foreign DNA, recombinant PPV are identified by:
a. detecting the foreign DNA, e.g. by means of DNA/DNA hybridizations
b. amplifying the foreign DNA by means of PCR
c. expressing the foreign DNA with the aid of recombinant viruses
With regard to a.
For this, the DNA is isolated from the virus in question and hybridized with nucleic acid which is at least in parts identical to the inserted foreign DNA.
The PPV which have been single-plaque purified and which have been identified as recombinant are preferably tested once again for the presence and/or expression of the foreign DNA. Recombinant PPV which stably contain and/or express the foreign DNA are available for further use.
With regard to c.
Expression of the foreign DNA can be detected at the protein level by, for example, infecting cells with a virus and then carrying out an immunofluorescence analysis using specific antibodies against the protein encoded by the foreign DNA, or by carrying out an immunoprecipitation or a Western blotting using antibodies against the protein encoded by the foreign DNA using the lysates of infected cells.
Expression of the foreign DNA can be detected at the RNA level by identifying specific transcripts. For this, RNA is isolated from Virus-infected cells and hybridized with a DNA probe which is at least in parts identical to the inserted foreign DNA.